The N-Terminally Truncated µ3 and µ3-Like Opioid Receptors Are Transcribed from a Novel Promoter Upstream of Exon 2 in the Human OPRM1 Gene

The human µ opioid receptor gene, OPRM1, produces a multitude of alternatively spliced transcripts encoding full-length or truncated receptor variants with distinct pharmacological properties. The majority of these transcripts are transcribed from the main promoter upstream of exon 1, or from alternate promoters associated with exons 11 and 13. Two distinct transcripts encoding six transmembrane domain (6TM) hMOR receptors, µ3 and µ3-like, have been reported, both starting with the first nucleotide in exon 2. However, no mechanism explaining their initiation at exon 2 has been presented. Here we have used RT-PCR with RNA from human brain tissues to demonstrate that the µ3 and µ3-like transcripts contain nucleotide sequences from the intron 1-exon 2 boundary and are transcribed from a novel promoter located upstream of exon 2. Reporter gene assays confirmed the ability of the novel promoter to drive transcription in human cells, albeit at low levels. We also report the identification of a “full-length” seven transmembrane domain (7TM) version of µ3, hMOR-1A2, which also contains exon 1, and a novel transcript, hMOR-1Y2, with the potential to encode the previously reported hMOR-1Y receptor, but with exon Y spliced to exon 4 instead of exon 5 as in hMOR-1Y. Heterologous expression of GFP-tagged hMOR variants in HEK 293 cells showed that both 6TM receptors were retained in the intracellular compartment and were unresponsive to exogenous opioid exposure as assessed by their ability to redistribute or affect cellular cAMP production, or to promote intracellular Ca2+ release. Co-staining with an antibody specific for endoplasmic reticulum (ER) indicated that the µ3-like receptor was retained at the ER after synthesis. 7TM receptors hMOR-1A2 and hMOR-1Y2 resided in the plasma membrane, and were responsive to opioids. Notably, hMOR-1A2 exhibits novel functional properties in that it did not internalize in response to the opioid peptide [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO).© 2013 Andersen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Schematic presentation of <i>OPRM1</i> gene structure and alternative splicing.

<p>Exons and introns are shown by boxes and horizontal lines, respectively. The approximate sizes of introns (in kb) are indicated. The 5′ extension of exon 2 and the new exon 3B are indicated by grey boxes. The locations of the promoters upstream of exons 11, 1, 13 and 2 are indicated. The transcriptional start sites are indicated by arrows. Putative translation start and stop codons are indicated by open and filled triangles, respectively. For hMOR-1A, the figure includes the full length sequence of hMOR-1A (GenBank accession number NM_001008504.2) which has a longer 3′ UTR than the originally published sequence. This extended sequence was confirmed in the present study (data not shown). All references are commented upon elsewhere in the manuscript, with the exception of the work by Du <i>et al.. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071024#pone.0071024-Du1" target="_blank">[32]</a> and Choi <i>et al.. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071024#pone.0071024-Choi1" target="_blank">[33]</a>. ^a The full length sequence of hMOR-1AΔ (µ3-like) was deposited in GenBank by Baar <i>et al</i>. in 2003 as “MOR-1W” (Genbank accession no. AY364890).</p

The N-Terminally Truncated µ3 and µ3-Like Opioid Receptors Are Transcribed from a Novel Promoter Upstream of Exon 2 in the Human <i>OPRM1</i> Gene

<div><p>The human µ opioid receptor gene, <i>OPRM1</i>, produces a multitude of alternatively spliced transcripts encoding full-length or truncated receptor variants with distinct pharmacological properties. The majority of these transcripts are transcribed from the main promoter upstream of exon 1, or from alternate promoters associated with exons 11 and 13. Two distinct transcripts encoding six transmembrane domain (6TM) hMOR receptors, µ3 and µ3-like, have been reported, both starting with the first nucleotide in exon 2. However, no mechanism explaining their initiation at exon 2 has been presented. Here we have used RT-PCR with RNA from human brain tissues to demonstrate that the µ3 and µ3-like transcripts contain nucleotide sequences from the intron 1-exon 2 boundary and are transcribed from a novel promoter located upstream of exon 2. Reporter gene assays confirmed the ability of the novel promoter to drive transcription in human cells, albeit at low levels. We also report the identification of a “full-length” seven transmembrane domain (7TM) version of µ3, hMOR-1A2, which also contains exon 1, and a novel transcript, hMOR-1Y2, with the potential to encode the previously reported hMOR-1Y receptor, but with exon Y spliced to exon 4 instead of exon 5 as in hMOR-1Y. Heterologous expression of GFP-tagged hMOR variants in HEK 293 cells showed that both 6TM receptors were retained in the intracellular compartment and were unresponsive to exogenous opioid exposure as assessed by their ability to redistribute or affect cellular cAMP production, or to promote intracellular Ca²⁺ release. Co-staining with an antibody specific for endoplasmic reticulum (ER) indicated that the µ3-like receptor was retained at the ER after synthesis. 7TM receptors hMOR-1A2 and hMOR-1Y2 resided in the plasma membrane, and were responsive to opioids. Notably, hMOR-1A2 exhibits novel functional properties in that it did not internalize in response to the opioid peptide [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO).</p></div

Amplification of the hMOR-1AΔ splice variant originating from the novel E2 promoter.

<p><b>A.</b> RNA from human thalamus was used in RT-PCR with forward primers from intron 1 (Int1_F2 and nested Int1_a) and reverse primers from exon 3C (3C_a and nested 3C_b). <b>B.</b> The resulting PCR product had a size of about 1400 bp, confirming its origin from cDNA and not genomic DNA (a product originating from genomic DNA would have a predicted size of 2200 bp). <b>C.</b> Sequences flanking the intron1-exon2 border. The intron 1 sequence is indicated in lowercase letters and the exon 2 sequence in uppercase letters. The forward primers from intron 1 used in RT-PCR are indicated in bold and are underlined. The putative potential translation start codon (ATG) is indicated in bold.</p

PCR primers.

a<p>: S, sense; A, antisense; ^b: numbering according to GenBank Accession no. NG_021208.</p><p>Restriction sites in Int1_BglII-4 and Int1_XmaI-4 primers are underlined.</p

Distribution patterns of C-terminally GFP-tagged hMOR-1 variants transiently expressed in HEK 293 cells.

<p>HEK 293 cells were transiently transfected with expression constructs for hMOR-1, hMOR-1A, hMOR-1AΔ (µ3-like), hMOR-1A2, µ3 and hMOR-1Y2 as indicated. In untreated cells (t = 0), hMOR-1, hMOR-1A and hMOR-1Y2 receptors were localized predominantly in the plasma membrane, whereas the novel variant hMOR-1A2 was observed both in the plasma membrane and in intracellular granula. The truncated splice variants hMOR-1AΔ (µ3-like) and µ3 were retained largely intracellularly, either spread throughout the entire cell (like most cells expressing µ3) or associated with intracellular structures and excluded from the nucleus as for many of the cells expressing hMOR-1AΔ (µ3-like). In cells exposed to DAMGO (<b>A</b>), hMOR-1, hMOR-1A and to some extent hMOR-1Y2 receptors were observed in numerous intracellular vesicles. No clear effect of DAMGO treatment (5 and 10 µM) was observed with the novel hMOR-1A2. For the truncated receptors (hMOR-1AΔ and µ3), exposure to DAMGO had no effect on receptor localization. Exposure to morphine (<b>B</b>) or M6G (<b>C</b>) did not affect the intracellular distribution of receptors. Fields of cells were analyzed by confocal microscopy as described in Materials and Methods.</p

hMOR-1AΔ is associated with endoplasmic reticulum.

<p>Transiently transfected HEK 293 cells expressing GFP-tagged hMOR-1AΔ (µ3-like) were fixed in paraformaldehyde and stained with an antibody against endoplasmic reticulum. The staining pattern caused by antibody (<b>A</b>) and the receptor itself (<b>B</b>) demonstrates a high degree of overlap, shown as yellow color in <b>C</b>. This strongly indicates that in cells with this staining pattern, hMOR-1AΔ (µ3-like) is associated with the endoplasmic reticulum.</p

Inhibition of forskolin-stimulated cAMP accumulation by opioids in hMOR-1 variants^a.

a<p>The IC₅₀ and maximal inhibition were calculated by nonlinear regression analysis using Prism 4.0, GraphPad Software. Results are means ± S.E.M. of 3–7 independent determinations. <b>Maximal inhibition</b>: Maximal inhibition was determined after correcting for different receptor expression levels by western blot analyses (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071024#pone.0071024.s003" target="_blank">Figure S3</a>). The expression levels of receptor variants were calculated relative to that of hMOR-1 (which was set to 1). Significant differences in maximal inhibition were observed for DAMGO (p<0.05), morphine (p<0.01) and M6G (p<0.01). For maximal inhibition by DAMGO, post hoc Tukey analyses showed that hMOR-1 differed from hMOR-1A (p<0.05). For maximal inhibition by morphine, post hoc Tukey analysis showed that hMOR-1 differed from hMOR-1A2 (p<0.01) and hMOR-1A (p<0.05). For maximal inhibition by M6G, post hoc Tukey analysis showed that hMOR-1 differed from hMOR-1A2 (p<0.05) and hMOR-1A (p<0.05). <b>IC₅₀</b>: Significant differences of IC₅₀ were observed for DAMGO (p<0.0001), morphine (p<0.05) and M6G (p<0.05). For DAMGO, post hoc Tukey analyses showed that the IC₅₀ value of hMOR-1 differed from hMOR-1A2 (p<0.001), and hMOR-1A2 differed also from hMOR-1A (p<0.001) and hMOR-1Y2 (p<0.001). For morphine, post hoc Tukey analyses showed that the IC₅₀ value of hMOR-1 differed from hMOR-1A2 (p<0.05). For M6G, post hoc Tukey analyses showed that the IC₅₀ value of hMOR-1 differed from hMOR-1A2 (p<0.05) and hMOR-1A2 differed also from hMOR-1A (p<0.05) and hMOR-1Y2 (p<0.05). ND: not determinable.</p

Structure and activity of the E2 promoter.

<p><b>A.</b> Schematic presentation of the E2 promoter. Putative transcription factor binding elements are indicated by triangles. The position of an Alu repeat element is indicated by a horizontal arrow. The promoter sequence was examined by the MatInspector and PATCH™ public 1.0 programs for potential binding sites for known transcription factors. <b>B.</b> Activity of the E2 promoter assessed by reporter gene assay. HeLa, SH-SY5Y and BE(2)-C cells were transfected with empty pGL3 Basic vector (Promega) or pGL3 containing the putative promoter region, as well as the control vector pRL-TK. Luciferase activity was measured using a dual luciferase kit according to the manufacturer's instructions (Promega). The measurements were performed in two independent experiments with three to six parallels in each. Each experiment comprised three different cell lines and showed comparable results. The results from one representative experiment are shown. The luciferase activity was significantly higher in all cell lines transfected with the novel E2 promoter region as compared to pGL3 Basic vector (p<0.005, student's t-test).</p

P-selectin genotype is associated with the development of cancer cachexia

The variable predisposition to cachexia may, in part, be due to the interaction of host genotype. We analyzed 129 single nucleotide polymorphisms (SNPs) in 80 genes for association with cachexia based on degree of weight loss (>5, >10, >15%) as well as weight loss in the presence of systemic inflammation (C‐reactive protein, >10 mg/l). 775 cancer patients were studied with a validation association study performed on an independently recruited cohort (n = 101) of cancer patients. The C allele (minor allele frequency 10.7%) of the rs6136 (SELP) SNP was found to be associated with weight loss >10% both in the discovery study (odds ratio (OR) 0.52; 95% confidence intervals (CI), 0.29–0.93; p = 0.026) and the validation study (OR 0.09, 95% CI 0.01–0.98, p = 0.035). In separate studies, induction of muscle atrophy gene expression was investigated using qPCR following either tumour‐induced cachexia in rats or intra‐peritoneal injection of lipopolysaccharide in mice. P‐selectin was found to be significantly upregulated in muscle in both models. Identification of P‐selectin as relevant in both animal models and in cachectic cancer patients supports this as a risk factor/potential mediator in cachexia